Indoor aeroallergens from American cockroaches and mites initiate atopic march via cutaneous contact in a murine model

The progression of allergic diseases from atopic dermatitis in childhood to other allergic conditions such as asthma in later life is often referred to as the atopic march. In order to study the relationship between cutaneous sensitization by aeroallergen and atopic march, we established a mouse model to test the hypothesis using American cockroaches and house dust mites as the model allergens. Mice were sensitized via skin with native cockroach extract (CraA) or recombinant Per a 2 and Der p 2 proteins without adjuvant. Each mouse was subjected to a total of three 1-week patching sensitizations with a 2-week interval in between each application. The resulting immunological variables in sera, scratching behavior, airway hyperresponsiveness (AHR), and pathology of skin lesions and nasal mucosa were evaluated. In mice, application of CraA, rPer a 2, and rDer p 2 aeroallergens through skin patching induced significantly high levels of both total IgE and specific IgEs. The epicutaneous sensitization after a subsequent allergen challenge showed a significant increase in scratch bouts, AHR, epidermal thickness, and eosinophil counts in the skin compared with the control mice. In addition, stimulation of murine splenocytes with allergens increased higher levels of Th2 cytokines, anti-inflammatory cytokines, and chemokines excretion. Our study provides evidence supporting that epicutaneous sensitization to aeroallergens also led to nasal and airway symptoms comparable to atopic march as described in humans. We hope this new allergy model will be useful in the development of new preventive and therapeutic strategies aimed at stopping the atopic march.


Introduction
The sequential development of allergic disease manifestations during early childhood is often termed the "atopic march". It describes a typical clinical feature of patients with atopic dermatitis (AD)/skin allergy symptoms in early childhood and subsequently develops allergic rhinitis and asthma/airway allergies [1][2][3][4]. There is increasing evidence that other factors influence the subsequent progression of airway allergies, allergic rhinitis, and allergic asthma later in life [5- 8]. As the most common chronic inflammatory skin disease, AD has a lifetime prevalence in US school-age children of up to 18% [9] and affects 6.7% of the general population in Taiwan [10]. Though it more frequently occurs in childhood, it can affect individuals at any age. Dermal sensitization of food allergens through defective skin has been studied in several murine models. Exposing mice to ovalbumin (OVA) or peanut on abraded skin leads to significant specific IgE responses [11]. Topical application with OVA in mice with loss-of-function mutations in the filaggrin (FLG) gene resulted in cutaneous inflammation and sensitization, as measured by specific IgE to OVA, but not in wild-type mice [12]. Benor et al. demonstrated that cutaneous exposure to peanut oil induces systemic and pulmonary peanut hypersensitivity reactions in a mouse model [13]. In a prospective birth cohort study, Lack and colleagues found that low-dose exposure to peanut oil applied to infants with inflamed skin was associated with an increased risk of peanut allergy at an early age [14]. Studies by Brough and colleagues also discovered that peanut allergens in household dust may promote peanut sensitization and eczema severity [15,16]. This impact was even more significant in children with filaggrin loss-of-function mutation [17].
Although this dermal sensitization of food allergen via defected skin can be recapitulated in mice, however, there has been a lack of suitable animal models to study cutaneous sensitization to aeroallergens and subsequent development of AD and airway allergies. We hypothesized that aeroallergens could sensitize the host via cutaneous contact, initiating the atopic march.
We established a mouse model to test the hypothesis that atopic march can be triggered by contact with aeroallergens via skin using the leading indoor allergens of American cockroaches and house dust mites as the model allergens.

Preparation of endotoxin removal American cockroach extract and recombinant Per a 2 and Der p 2 proteins
American cockroach extract (CraA) was prepared from P. americana with Coca's solution, as described previously [18]. E. coli-expressed American cockroach major allergen Per a 2 and house dust mite major allergen Der p 2 recombinant proteins (rPer a 2 and rDer p 2) were purified by rapid affinity column chromatography (Novagen, Darmstadt, Germany) [19]. The CraA and both of recombinant proteins were further purified by Endotoxin Detoxi-Gel (Pierce, Illinois, USA) and sterilized by passing through a 0.22-μm syringe filter (Millipore, Billerica, MA, USA). Finally, the protein concentration was determined by the Coomassie brilliant G-250 protein-dye binding method of Bradford using bovine serum albumin as a standard according to the manufacturer's procedures (Bio-Rad, Hercules, CA, USA).

Experimental design of allergen-induced atopic march in a murine model
Six-week-old female BALB/c mice from the National Laboratory Animal Center, Taiwan, were used for the experiments. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Taichung Veterans General Hospital (La-1091738). The scheme of the experimental design is shown in the

Epicutaneous sensitization and intradermal/intranasal challenge
To induce AD-like skin lesions, 50 μg each of CraA, rPer a 2 or rDer p 2 in 100 μl of phosphate-buffered saline (PBS) were applied on 1cm 2 gauze pads. The patches were placed on the shaved back skin with a transparent dressing (3M HealthCare) for one week and then removed. Two weeks later, an identical patch was reapplied onto the same skin site. Each mouse received a total of three 1-week exposures at 2-week intervals. To check the sensitization response, skin scratching behavior and airway hyper-responsiveness (AHR) were performed on days 55 and 56 after intradermal (ID) and intranasal (IN) challenges, respectively. Control mice received PBS for both patching sensitization and challenge experiments. Serum samples were collected from the submandibular vein bi-weekly and stored at -20˚C until analysis. All mice were sacrificed on day 57, and tissues from skin, nose, and spleen were collected for further study.

Scratching behavior
After the 3 cycles of allergen patching, the scratching behaviors were videotaped for 1 h immediately following the last ID challenge at the right leg dorsal region on day 55. Each scratching event around the ID site was recorded using video playback [20].

Measurement of airway hyperresponsiveness (AHR)
On day 56, the AHR of mice was measured using a whole-body Buxco mouse plethysmograph (Buxco, NY, USA) 24 h after the last IN challenge. Mice were placed in the main chamber and challenged with aerosolized methacholine at 0, 25, and 50 mg/mL concentrations generated by a nebulizer (Buxco aerosol distribution system). The degree of bronchoconstriction was measured and averaged for 3 min after each nebulization. Data were expressed as enhanced pause (Penh) by the following equation: Penh = pause × (PEP/PIP). Pause, PEP, and PIP refer to the expiration time, peak expiratory pressure, and peak respiratory pressure, respectively.

Histological analysis of skin lesions and nasal mucosal tissue
The mice were sacrificed 48 h after the last allergen challenge, and the dorsal patched skins were removed immediately. The excised skin specimens were cut into two pieces. One of the skin lesions was immediately submerged in RNA stabilization solution (Invitrogen, CA, USA) for RNA extraction. The other piece was fixed in 10% neutral formalin overnight and embedded in paraffin. For nasal mucosal evaluation, the mice were decapitated, and then the heads were fixed in 10% neutral formalin for 72 h. The heads were then washed in running water and placed in Calci-Clear Rapid for decalcification for 72 h. The paraffin blocks of skin and nasal tissues were divided into 4-μm thick sections and stained with hematoxylin and eosin (H&E). All slides were examined under a Hamamatsu NanoZoomer 2.0 HT slide scanner (Hamamatsu, Japan) at 400-fold magnification.

Analysis of total IgE, allergen-specific IgE, IgG1, and IgG2a antibodies by ELISA
According to the manufacturer's instructions, the total IgE levels were measured using an IgE mouse ELISA kit (Invitrogen). Ninety-six-well MaxiSorp plates (Nunc, Denmark) were coated overnight at 4˚C with a 5 μg/mL monoclonal antibody. All of the following steps were performed at room temperature. After washing twice with PBS/0.05% Tween 20, the plates were blocked with PBS plus 1% BSA for 2 h and then incubated for 2 h with prediluted serum (1:10) or 2-fold serial dilutions of mouse IgE standards. After washing, the detection antibody (1:250) was added and incubated for 2 h. The plates were washed followed by addition of streptavidinhorseradish peroxidase conjugate (1:400) for 30 min. For visualization, tetramethylbenzidine (TMB) substrate was added, and the reaction was stopped with 1 M phosphoric acid. The absorbance was measured at 450 nm using an ELISA reader (TECAN, Austria).

RNA preparation and quantitative real-time PCR
Total RNA was extracted from the lungs, skin lesions, and stimulated splenocytes using TRI-ZOL Reagent (Invitrogen, Carlsbad, CA). RNA concentrations and the 260/280 nm absorbance ratio were determined using NanoDrop One ND1000 (Thermo; Waltham, MA, USA). cDNA was reverse-transcribed using a SuperScript III kit (Invitrogen) according to the manufacturer's protocol using 1 μg total RNA in 20 μL reactions. The quantitative real-time PCR was performed on a StepOnePlus TM system (Applied Biosystems, CA, USA) with primers listed in S1 Table. The gene expression data were expressed as a fold increased between untreated cells and allergen-stimulated cells after normalized with the housekeeping gene βactin.

Statistical analysis
Statistical analysis was performed using IBM SPSS software, version 22 (IBM Corporation, Armonk, NY, USA), with appropriate methods. P-values less than 0.05 were considered to be statistically significant.

Detection of total IgE and allergen-specific IgE, -IgG1, and -IgG2a in sera after epicutaneous allergen exposure
To establish epicutaneous sensitization using purified allergens, we patched mice with cockroach crude extract (CraA), purified rPer a 2, or rDer p 2 recombinant proteins without adding an adjuvant. Quantitative analysis of total IgE was performed to evaluate the sensitized status of the mice. Among the four groups, mice that were patched with CraA, rPer a 2 and rDer p 2 developed significantly higher levels of total IgE at week 8 (Fig 2A) in comparing with the PBS control group.
We further examined allergen-specific serum antibodies before and after skin exposure by in-house ELISA. After patching, CraA-and rPer a 2-specific IgE antibodies were significantly increased at two weeks following sensitization. The rDer p 2-specific IgE level was not significantly elevated until the fourth week (Fig 2B). Only rPer a 2 group showed a marked elevation of allergen-specific IgG1 level in a time-dependent manner. rDer p 2-specific IgG1 was significantly increased at week 8 ( Fig 2C). However, our model revealed that Per a 2-specific IgE rose significantly in the early stage and reduced gradually to the basal level at week 8. A similar antibody responsive pattern was also observed in specific IgG2a (Fig 2D).

Measurement of skin hypersensitivity by scratch counts, cytokine expression, and histopathology after allergen challenge
AD is a chronic skin disorder that is characterized clinically by intense itching and scratching, leading due to allergic inflammation. To examine whether re-exposure to aeroallergens would elicit itching after epicutaneous sensitization, the scratching events were counted from video playback. Fig 3A shows that the mice in the CraA, rPer a 2, and rDer p 2 groups exhibited significantly increased scratching bouts after the allergen challenge compared to the control mice.
We measured cytokines that are known to participate in the progression of AD. IL-13 is one of the major pathogenic cytokines released in response to cutaneous allergens in AD [21]. IL-31 is associated with cutaneous lymphocyte antigen-positive skin-homing T cells and itching in AD patients [22]. Thymic stromal lymphopoietin (TSLP) is a critical pro-inflammatory cytokine in both acute and chronic skin lesions of AD [23]. As shown in Fig 3B, 3C and 3D, mice with rPer a 2-patch exhibited a marked increase in mRNA expressions of IL-13, IL-31, and TSLP in comparing with PBS controls. The rDer p 2-patched mice showed the most significant increase in IL-13 and IL-31 in the skin lesions, while TSLP production was found significantly increased in American cockroach-sensitized groups, CraA and rPer a 2.
Conversely, skin lesions of AD are characterized by increased skin thickness and dermal infiltration of inflammatory cells. Histological analysis was performed to determine whether aeroallergen-painting sensitization induced immune cell infiltration in AD-like skin lesions. H&E staining showed epidermal hyperplasia and accumulation of inflammatory cells in the dermis of the allergen-patched groups compared to the PBS group (Fig 4A). Repeated patching with either native or recombinant allergens on the skin was able to induce significant epidermal thickness (Fig 4B). Moreover, all of the aeroallergen-treated groups showed a significant increase in total cells, eosinophil, and neutrophil counts compared to the PBS control group (Fig 4C).

Measurement of allergic nasal inflammation by histopathology after allergen challenge
We then examined whether epicutaneous application of allergens could induce rhinitis-like inflammation in the nose. In the CraA, rPer a 2, and rDer p 2 mice groups after IN challenge, there were mild tissue inflammations with eosinophilia in both the nasal and maxilla turbinates ( Fig 5A) but not in the control group.

Measurement of airway hyper-responsiveness by plethysmograph and pulmonary inflammation by H&E staining
To examine whether or not this epicutaneous exposure of aeroallergens-induced AD mice may lead to allergic asthma, mice underwent IN challenge with specific allergens for each

PLOS ONE
group. One day after the last challenge, mice were treated with saline aerosol followed by increasing concentrations of methacholine aerosol, and Penh was calculated. In all three sensitized groups, mice showed significantly increased Penh upon methacholine exposure, but not in the PBS group ( Fig 5B).
Additionally, histopathologic features of the murine lungs are shown in Fig 5C. The examination of the lung tissues from all three sensitized groups reveals a significant increase in cellular infiltration of total inflammatory cells and eosinophils compared with the PBS group.

Discussion
This study used indoor aeroallergens from cockroaches and mites as the model allergens to determine the role of aeroallergens in initiating atopic march via skin. It is well known in respiratory allergy that inhaled sensitization to cockroach and mite allergens plays a critical role in asthma. However, little is known about the initiation and activation of immunity by cockroaches and mite allergens via the skin. In the present study, we found that, in addition to the conventional airway exposure and sensitization, indoor aeroallergens from cockroaches and mites do have the ability to initiate allergic inflammation of the skin as well as nose and lung via cutaneous exposure.
It has been reported that epicutaneous application of house dust mite induced allergic inflammation in non-lesional skin of patients with atopic dermatitis and systemic allergic inflammation in a canine atopic model [24,25]. Our previous studies have shown that different components of cockroach allergens have different allergenicity [26,27]. We found that sensitization to Per a 2 of the American cockroach correlates with greater clinical severity among airway allergic patients in Taiwan compared to other allergen components of cockroaches [26]. Per a 2 is a 42 kDa protein with aspartic protease-like biochemical activity [28]. It is abundant

PLOS ONE
in cockroach feces and is very stable in the household environment. It remains undegraded after a year of decomposition compared to other cockroach allergen components [27,29]. In this study, 50 μg of Per a 2 induced an earlier increase of specific-IgE on week 2 and highest IgG1 level on week 4 than the other same dose tested allergens in initiating allergic inflammation via skin. In general, allergen-specific IgE actives both mast cells and basophils by binding to Fc�RI receptor on their surface; however, these cells also express IgG Fc receptor, FcγRIIb, that suppress their IgE-mediated activation. Previous studies showed that sensitization in mice induced both IgE and IgG1 responses [30]. The results echo our previous observations of airway allergy [26].
Allergen exposure is a determinant of atopic march onset. However, it remains controversial whether high or low-dose exposure is more likely to result in allergy (29). It has been reported that a high dose (120 μg/mouse) of cutaneous mite allergen exposure may induce IgG-mediated protection against anaphylaxis, even accompanied by IgE production [31]. However, low-dose exposure to cutaneous cockroaches and mite allergens in this study seemed to induce the allergen-specific IgE and the triad of skin, nose, and airway allergies. Further studies are required to establish whether high-dose exposure can induce more protective Per a 2-specific IgG2a at eight weeks to "neutralize" Per a 2-specific IgE, as shown in Fig 2. There were some limitations in the current study. First, in order to control the dose of the allergen exposure, we patched the mice for 3 one-week exposures, with two-week interval in between each exposure. In the real world, patients may be exposed to these indoor allergens in a more continuous and persistent pattern. Whether these differences could change the results of allergic inflammation requires further study. Secondly, in our mouse model, the allergic inflammation of the skin, nose, and lung was observed simultaneously after epicutaneous aeroallergen exposure. In the current study, we did not provide a comprehensive depiction of the sequential development of allergic diseases in the atopic march. Our mouse model developed patterns of multimorbidity after skin sensitization with strong allergens which partly supports  [32]. Thus, in agreement with Yu's discussion [33], the sequence of each allergic disease that occurred in the atopic march progression still remained unclear. However, our in vivo evidence did demonstrate that skin exposure to indoor aeroallergens can initiate systemic allergic inflammation in the skin, nose, and lungs. The findings also support the crucial importance of the use of skin emulsions to ameliorate skin barrier dysfunction for preventing or delaying the initiation of atopic march. Finally, in our study, the negative control group received PBS for both sensitization and challenge for comparison. For immune response study, using a non-allergic protein instead of PBS may be a much better alternative.
In conclusion, the current study provides additional evidence supporting the outside-in hypothesis of the development of atopic march. We hope this new allergy model will be useful in the development of new preventive and therapeutic strategies aimed at stopping the atopic march. Mouse splenocytes were cultured under stimulation with individual allergen for 3 days and total RNA were extracted for cytokine expression. Quantitative PCR was performed to determine the mRNA expression of IL4, IL-5, IL-9, IL-13, IFN-γ, TNF-α, IL-6, IL-10 and MCP-1. Fold increase is shown as mean±SEM with respect to untreated cells after correction by the house-keeping gene β-actin. Statistical differences between PBS-and allergen-patching groups were assessed by an independent 2-tailed t test. *p<0.05, ** p<0.01, *** p<0.001.